Antiretroviral therapy (ART) is widely used and subjects on ART were reported to develop antibodies against these drugs when used as monotherapy. The aim of this study was to determine the proportion of HIV-sero positive subjects on antiretroviral therapy who developed antibodies to the drugs used as combination therapy and to associate the presence of antibodies to CD4 cell count which is a marker of disease progression. A total of 300 subjects were randomly selected for the study, consisting of 100 HIV-sero positive subjects already on ART, 100 newly diagnosed HIV-sero positive ART- naive subjects and 100 HIV-sero negative individuals who were monitored as controls. CD4 cell count and complete blood count were assayed using BD FASCOUNT CD3/CD4 and Sysmex KX-21N autoanalyzer respectively. Antigen-Antibody agglutination technique was used to detect antibodies produced against antiretroviral drugs in HIV -sero positive subjects on ART. The results showed that antibodies to antiretroviral drugs (tenofovir disoproxil fumarate/lamivudine Efavirenz + lamivudine/ nevirapine/zidovudine combination) were detected in 33(33%) out of the 100 HIV-sero positive subjects on ART studied. Significantly higher CD4 cell count (p&lt;0.001), PCV (p&lt;0.001) and white blood cell count (p&lt;0.001) were recorded among control group than HIV positive subjects whether they in 33(33%) out of the 100 HIV- sero positive subjects on ART combination studied.&nbsp; The use of combination therapy has led to a smaller number of subjects on ART who developed antibody as against a higher percentage on monotherapy that was previously reported. Significantly lower level of platelet was observed among HIV-sero positive subjects who developed ART antibodies.&nbsp

Emokpae, Mathias Abiodun

Obazelu, Progress Arhenrhen

English

AJOL - African Journals Online

SJMLS - 7(1) - 013Association of CD4 cell count and antiretroviral therapy antibody production in Human Immunodeficiency virus seropositive subjects on antiretroviral drugs in Benin City, Nigeria.1 1Progress Arhenrhen OBAZELU*  and Mathias Abiodun EMOKPAE Department of Medical Laboratory Science, School of Basic Medical Sciences, University of Benin.Author for Correspondence*: progress.obazelu@uniben.edu/+234-805-673-3255/0000-0002-4492-5684. https://dx.doi.org/10.4314/sjmls.v7i1.13 Sokoto Journal of Medical Laboratory Science 2022; 7(1): 101 - 106SJMLSAbstract in 33(33%) out of the 100 HIV- sero positive Antiretroviral therapy (ART) is widely used and subjects on ART combination studied.  The use subjects on ART were reported to develop of combination therapy has led to a smaller antibodies against these drugs when used as number of subjects on ART who developed monotherapy. The aim of this study was to antibody as against a higher percentage on determine the proportion of HIV-sero positive monotherapy that was previously reported. subjects on antiretroviral therapy who developed Significantly lower level of platelet was antibodies to the drugs used as combination observed among HIV-sero positive subjects who therapy and to associate the presence of developed ART antibodies.antibodies to CD4 cell count which is a marker of disease progression. A total of 300 subjects were Keywords: ART antibodies, HIV, CD4 cell randomly selected for the study, consisting of count, Haematological parameters.100 HIV-sero positive subjects already on ART,   100 newly diagnosed HIV-sero positive ART- Introductionnaive subjects and 100 HIV-sero negative Antiretroviral therapy (ART) involving the use individuals who were monitored as controls. of a combination of antiretroviral drugs made up CD4 cell count and complete blood count were of two nucleoside reverse transcriptase assayed using BD FASCOUNT CD3/CD4 and inhibitors (NRTI) and an important non-Sysmex KX-21N autoanalyzer respectively. nucleoside reverse transcriptase inhibitor Antigen-Antibody agglutination technique was (NNRTI) or protease inhibitors (PI) has become used to detect antibodies produced against the gold standard in the management of patients antiretroviral drugs in HIV -sero positive with HIV infection unlike the use of subjects on ART. The results showed that monotherapy which was the practice many years antibodies to antiretroviral drugs (tenofovir ago (Odunukwe et al., 2005).  According to disoproxil fumarate/lamivudine Efavirenz + Andrade and Cotter (2006), disorders like l a m i v u d i n e /  n e v i r a p i n e / z i d o v u d i n e  abnormalities in lipid metabolism lipodystrophy, combination) were detected in 33(33%) out of insulin resistance have been associated with the 100 HIV-sero positive subjects on ART protease inhibitors. This has led to a decline in studied. Significantly higher CD4 cell count the use of protease inhibitors as antiretroviral (p<0.001), PCV (p<0.001) and white blood cell therapy. Antiretroviral therapy has been reported count (p<0.001) were recorded among control to improve packed cell volume of patients group than HIV positive subjects whether they infected with   HIV, though there are evidences were on ART or not. However, a significantly that these drugs have cytopenic effect when used lower level of platelet was observed in HIV-sero as monotherapy (Odunukwe et al., 2005). The positive subjects on ART who developed use of ART has led to decreased mortality of antibodies. The findings from this study showed infected subjects since it was introduced in 1996 that antibody to antiretroviral drugs was detected (Gea-banacloche et al., 1999).SJMLS Volume 7, Number 1 March, 2022    Page 101It was reported that numerous individuals living Sample collection and analysis: Ten with HIV that were on ART treatment still millimeters of blood was obtained for analysis developed anaemia (Mildvan,  2003).  from each subject.  Five (5) milliliters of blood Antiretroviral therapy treatment was reported as were dispensed into Ethylene Diamine Tetra one of the causes of anaemia in HIV infected acetic acid (EDTA) (0.1ml of EDTA solution to subjects (Moyle, 2004). The antiretroviral drugs 5mls of blood sample) specimen container, cause anaemia by inhibition of haemoglobin mixed and analyzed immediately while the synthesis (Wood et al., 2002) and by production remaining blood sample was dispensed into of antibodies against the drugs or their plain specimen container. The sample in the plain metabolites (Omoregie et al., 2008). It was container was allowed to clot, and the serum previously reported that 81% HIV-sero positive separated into a pre-labeled plain container, the osubjects on ART had antibodies to one or more of serum was stored frozen at -20 C and was only the ART drugs when used as monotherapy thawed when needed for analysis. The measured (Omoregie et al., 2008). haematological parameters included: packed cell volume (PCV), haemoglobin concentration, The use of multiple drug therapy has been shown lymphocyte count, platelet count and white to be more effective in the management of blood cell count (WBC). Analysis for full blood patients with HIV infection (Barlett et al., 2006; count was carried out using the Sysmex KX-21N Thompson, 2010). The proportion of individuals auto analyzer (Sysmex Corporation Kobe, who developed antibodies to ART monotherapy Japan).  CD4 cell count was estimated using the was reported in Benin City, Nigeria (Omoregie et BD FASCOUNT CD3/CD4 auto analyzer (BD al., 2008). But no study to the best of our Biosciences, California, USA).                       knowledge has been conducted to evaluate                           an t ibody  deve lopment  aga ins t  ART-  Detection of Antibodies to ART Drugs  combination drug therapy. The objective of this Antigen-antibody agglutination method study therefore was to evaluate the proportion of demonstrated by Petz was used (Petz, 1986). A the HIV-sero positive subjects on ART- tablet of Tenofovir Disoproxil Fumarate/ combination therapy who developed antibodies lamivudine /Efavirenz and lamivudine/ to the drugs and also to associate the presence of nevirapine/zidovudine were dissolved in 10 these  ant ibodies  wi th  the  measured  milliliters of normal saline in a test tube, the drug immunological and haematological parameters. suspension was centrifuged. Equal volumes of group O washed cells (3%) and solution of Materials and Methods Tenofovir Disoproxil Fumarate/lamivudine Study parameters:                                          /Efavirenz and lamivudine/nevirapine/   A total of three hundred (300) individuals were zidovudine were placed in a test tube and then Oenrolled, consisting of 100 HIV-sero positive incubated at 37 C for 30 minutes. The drug-red subjects already on ART for 3 -6 months (24 blood cell mixture was washed four times to males and 76 females), 100 newly diagnosed remove excess drug. Equal volume of patient's HIV-sero positive naive subjects (27 males and serum and drug-red blood mixture was placed in a O73 females) 100 HIV-sero negative individuals clean test tube and incubated at 37 C for 1 hour. (volunteer donors consisting of 42 males and 58 The mixture was washed three times, Antihuman females) who served as controls, were recruited globulin (AHG) was added and centrifuged and for this study. The HIV positive individuals were haemolysis or agglutination was observed for. The attending HIV clinic at Central Hospital, Benin controls were similarly treated, ie City. Subjects who had physical manifestation of I. Drug red blood cell complex without full-blown AIDS were excluded from this study. patient's serum.II. Patient's serum and group O red blood cells Ethical consideration: The protocol for the without drug.study was reviewed and approved by the ethical III. Both results were negative.committee of Edo State Ministry of Health with reference number: HA.577/VOL.2/153.                                                                                                    SJMLSSJMLS Volume 7, Number 1 March, 2022    Page 102who developed HAART antibodies than their Statistical Analysiscounterparts who did not develop antibodies. The Results were expressed as mean±SEM and were differences in the mean lymphocyte count and Hb analyzed by Student's t-test. P-values of < 0.05 values between the two groups were not were considered statistically significant and statistically significant (table 1). Pearson correlation coefficient was calculated to determine the association between antibodies + The comparison of the values of measured produced and CD4  cell count.variables between HIV positive naïve, on ART and healthy HIV—negative controls are shown in Resultstable 2. The correlation of CD4 cell count with The results obtained in this study are shown in measured parameters among HIV subjects on Tables 1-4. A total of 33(33%) out of the 100 HIV-ART and controls showed that CD4 cell count sero positive subjects on ART-combination significantly correlated with lymphocyte count therapy had antibodies to the drugs used. The (R=0.526, p<0.001), PCV (R=0.279, p<0.01) and mean levels of CD4 cell count, platelets and white haemoglobin (R=0.309; p<0.05) in HIV-sero blood cell were significantly higher (p<0.001) in positive subjects on ART. Among the controls, HIV-sero positive subjects who developed CD4 count correlated positively with lymphocyte antibodies against HAART than those who did not (R=0.527, p<0.001), PCV (R=0.221, p<0.05) and develop antibodies. Conversely the mean level of hemoglobin (R=0.254, p<0.05).PCV was significantly lower (p<0.001) in subjects Table 1: Comparison of CD4 Cell Count and some Haematological parameters among HIV-Seropositive subjects in the absence or presence of ART antibodiesMeasured variables           Mean±SEM P-value   HIV-sero positive subjects on ART who developed antibodies  HIV-sero positive subjects on ART who did not develop antibodies   Number of subjects  CD4 (cell/µl)  33   561.33 ± 68.77  67  485.96 ± 36.36   <0.001  Platelets (x103/µl)  231.04 ± 14.07  213.26 ± 9.40 <0.001  WBC (x103/µl)  5.32 ± 0.22  5.21 ± 0.28 <0.001  Lymphocyte (x103/µl)  2.26 ± 0.21  2.02 ± 0.10 >0.05  PCV  (%)  29.63 ± 0.10  30.59 ± 0.53 <0.001  HB (g/L)  10.15 ± 0.37  10.56 ± 0.22 >0.05   Key: p≤0.05- Significant; p≥0.05- Not significantWBC: White Blood CellsPCV: Packed Cell VolumeHB: HaemoglobinSJMLSSJMLS Volume 7, Number 1 March, 2022    Page 103Table 2: Comparison of CD4 Cell Count and some haematological parameters of the studied populationMeasured variables                       Mean ± SEM                                                         P-value   ART naïve HIV Sero-positive HIV Sero-positive on ART HIV Sero-negative (controls)   Number of subjects  CD4 (cell/µl) 100  355.63b ± 28.14 100  503.52a ± 32.16 100  914.23c ± 25.41   <0.001  Platelets (x103/µl) 257.76b ± 11.52 217a.52 ± 7.90 206.10a ± 6.79 <0.001  WBC (x103/µl) 5.39a ± 0.23 5.25a ± 0.22 7.43b ± 0.18 <0.001  Lymphocyte (x103/µl) 2.19a ± 0.10 2.09a ± 0.09 1.96a ± 0.05 >0.05  PCV (%) 32.21b ± 0.67 30.59a ± 0.49 33.73b ± 0.34 <0.001  HB (g/dL) 11.04a ± 0.23 10.51a ± 0.19 10.96a ± 0.37 >0.05   Key: p≤0.05- Significant; p≥0.05- Not significantWBC: White Blood Cells; PCV: Packed Cell Volume; HB: HaemoglobinValues with different superscripts indicate significant different (ANOVA). Table 3: Correlation of CD4 cell count with studied parameters among HIV- sero positive subjects on ARTParameters                              CD4(Cell/µl)                     P-value  Platelet   (×103/µl)     -0.073                                  >0.05  WBC   (×103/µl)                               0.108                                    >0.05                Lymphocytes  (×103/µl)   0.526                                    <0.001                 PCV (%)   0.279                 <0.01                 HB(g/L)   0.309                                    <0.05                    Key: p≤0.05- Significant; p≥0.05- Not significantWBC: White Blood CellsPCV: Packed Cell VolumeHB: HaemoglobinSJMLSSJMLS Volume 7, Number 1 March, 2022    Page 104Table 4: Correlation of CD4 count with studied parameters among HIV-sero negative subjectsParameters                           CD4(Cell/µl)                 P-value              Key: p≤0.05- Significant; p≥0.05- Not significantWBC: White Blood CellsPCV: Packed Cell VolumeHB: Haemoglobin.Discussion naïve and control. This observation is at variance The presence of antibodies to tenofovir with previous study (Odunukwe et al., 2005) that disoproxil fumarate/lamivudine efavirenz and reported that HAART improved haematocrit lamivudine/ nevirapine/zidovudine were values. Our finding is however in agreement with a detected in 33(33%) out of the 100 HIV-sero previous report which reported a significantly positive subjects on ART combination therapy. higher PCV in the control group compared to The implication of this finding is that treatment subjects on HAART and HAART (Omoregie et of HIV- sero positive subjects with tenofovir al., 2008). The causes of anaemia infection are disoproxil fumarate/lamivudine Efavirenz and many and include bone marrow suppression by l a m i v u d i n e / n e v i r a p i n e / z i d o v u d i n e  HIV and HAART as haemoglobin synthesis was combinations has brought about a decrease in the reported to be inhibited by Zidovudine especially occurrence of ART- antibody development in Africans. Inadequate nutrition could be a major among subjects when compared with previous factor in HIV infection but was not considered in finding (Omoregie et al., 2008) which reported this study.that a total of 81(81%) out of the 100 HIV- sero positive subjects on ART developed antibodies The reasons for significantly higher levels of to ART monotherapy. Studies have shown that CD4 cell count, platelets and WBC in HIV the use of antiretroviral drugs may cause positive subjects who developed antibodies anaemia by producing antibodies against the compared with those who did not develop drugs or their metabolites. The mechanism could antibodies is not clear. The severity and be the attachment of the drugs or their progression of HIV infection could also metabolites to red blood cells surface which then influence anaemia. The CD4 cell count which is results in antibody production against the a measure of HIV infection progression antiretroviral drugs. The antibody will in turn correlated positively with PCV (r=0.279; attach to the red cell membrane. These sensitized p<0.01) and Hb (r=0.309; p<0.05).red cells either activates complement and are lysed or are damaged by the reticulo-endothelial Thrombocytopenia was reported among HIV system (Petz, 1986). This may had resulted in the subjects. Immune thrombocytopenic purpura significantly lower level of PCV between HIV- has been recognized as a diagnostic criterion of sero positive subjects on ART who developed AIDS related complex (Cooper and Merigan, antibodies compared to their counterparts who 1996).  Studies have shown that megakaryocytes did not develop antibodies. may be target for HIV infection as well as reservoir for the virus in the bone marrow, In this study, we observed a significantly lower suggesting a potential mechanism for defective level of PCV in subjects on HAART than HAART thrombocytopoiesis (Nubila et al., 2012). In this   Platelet (×103/µl)   0.044                      >0.05  WBC  (×103/µl)                  0.147                        >0.05     Lymphocytes  (×103/µl)  0.527                            < 0.001                  PCV (%)  0.221               <0.05                 HB(g/L)  0.254                            <0.05                   SJMLSSJMLS Volume 7, Number 1 March, 2022    Page 105study there was a significant decrease (p<0.001) Mildvan, D. (2003). Implication of anaemia in human immunodeficiency virus, cancer and in platelet count among the HIV-sero positive subjects. Furthermore, CD4 cell count hepatitis C virus. Clinical and Infectious significantly correlated with lymphocyte count Disease; 37: 293-296.(r=0.526, p<0.001) in HIV-sero positive subjects Moyle, G. (2004). Anaemia in persons with HIV on ART group. This finding also agrees with infection: prognostic marker and contributor previous study (Idowu et al., 2013). to morbidity. AIDS Review; 24: 13-18.Nubila, T., Ukaejiofo, E.O., Nubila, N. and Conclusion Okorie, G. (2012). Examination of        The findings from this study showed that haematoxicity of fixed-dose highly active antibody to antiretroviral drugs (tenofovir antiretroviral drug in Albino wistar rats. disoproxil fumarate/lamivudine Efavirenz + International Scholarly Research Notices; l a m i v u d i n e / n e v i r a p i n e / z i d o v u d i n e  10: 5402-5408.  combination) was detected in 33(33%) of the 100 Odunukwe, N., Idigbe, O., Karki, P., Adewole, T., HIV-sero positive subjects on ART. The number Onwujekwe, D., Audu, R. and Onyewuche, J. of subjects who developed antibody to (2005). Haematological and biochemical combination ART therapy was significantly response to treatment of HIV-1 infection with lower than previously reported. a combination of nevirapine + stavudine + lanivudine in Lagos, Nigeria. Turkish Journal References of Haematology; 22(3): 125-131.Andrade, A.C.O. and Cotter, B.R. (2006). Omoregie, R., Egbeobauwaye, A., Ogefere, H., Endothelial function and cardiovascular Omokaro, E.U. and Ekeh, C.C. (2008). Prevalence diseases in HIV infected patients. Brazilian of Antibodies to HAART Agents among HIV Journal of Infectious Diseases; 10: 139-145. Patients in Benin City, Nigeria. African Journal of Barlett, J.A., Faith, M.J., Demasi, R., Hermes, Biomedical Research; 11: 33-37.A., Quinn, J., Mondou, E. and Rousseau, F. Petz, L.D. (1986). Autoimmune and drug induced (2006). An updated systemic overview of immune haemolytic anaemia. In Rose, N.R. triple combination therapy in antiretroviral- Friedman, H. and Fahey, J. H. (Ed.) manual of rdnaïve HIV-infected adults. AIDS; 20(10): clinic laboratory immunology (3  edition). 2051-2064. American society for microbiology, Cooper, D.A. and Merigan, T.C. (1996). Clinical Washington D.C.  pp. 613-629.treatment. AIDS Supply; 10: 133-134. Thompson, M.A. (2010). Antiretroviral Gea-banacloche, J.C. and Lane, H.C. (1999). treatment of adult HIV infection: 2010 Immune reconstitution in HIV-1 infection. recommendations of the international AIDS AIDS; 13: 525-538. Society – USA panel. Journal of American Idowu, C.O., Oke, O.T., Afolayan, D.O., Medical Association; 304(3): 321-333. Olaniran, O.,  Akinloye, O.A. and  Wood, M.N., Spiegelman, D., Knox, T.A., Awodumila, O.O. (2013). Effect of HAART Forester, J.E., Connors, J.L., Skinner, S.C., on some haematological parameters, Silvia, M., Kin, J.H. and Gorbach, S.L. correlations between total lymphocyte and (2002). Nutrient in-take and body weight in a CD4 counts of HIV clients attending SSH, large HIV cohort that includes women and Ikole-Ekiti. International Journal of Biology minorities. Journal of American Dietician and Medical Research; 4(3): 3265-3270. Association; 102(2): 203-213.Citation: Progress Arhenrhen Obazelu and Mathias Abiodun Emokpae. Association of CD4 Cell Count and Antiretroviral Therapy Antibody Production in Human Immunodeficiency Virus Seropositive Subjects on Antiretroviral Drugs in Benin City, Nigeria. Sokoto Journal of Medical Laboratory Science; 7(1): 101 - 106.Copyright. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.SJMLSSJMLS Volume 7, Number 1 March, 2022    Page 106

Association of CD4 cell count and antiretroviral therapy antibody production in Human Immunodeficiency virus seropositive subjects on antiretroviral drugs in Benin City, Nigeria.

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Association of CD4 cell count and antiretroviral therapy antibody production in Human Immunodeficiency virus seropositive subjects on antiretroviral drugs in Benin City, Nigeria.

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